Mutations in cardiac myosin binding protein C (cMyBP-C) are a frequent cause of hypertrophic cardiomyopathy (HCM), a major cause of sudden cardiac death and heart failure. Mutations include single amino acid substitutions and premature stop codons, but it is unclear whether dominant negative effects of mutant proteins, depletion of wild-type protein due to an affected allele (haploinsufficiency), or aberrant protein processing/degradation leads to disease. To distinguish among these possibilities, we investigated the sarcomeric localization and functional effects of a spontaneous cMyBP-C missense mutation in Maine Coon cats, a naturally occurring feline model of HCM. Immunofluorescent localization using an antibody specific for the A31P mutation showed that A31P cMyBP-C was incorporated into the sarcomeres of cats heterozygous and homozygous for the A31P mutation with similar distribution patterns as wild-type cMyBP-C. However, dominant negative effects due to incorporation of the mutant protein were not evident because myofilament Ca2+ sensitivity of tension and rate of tension development were not different in permeabilized myocytes from wild-type versus A31P cats. Actin binding and in vitro motility experiments also showed no difference between wild-type and A31P recombinant feline C0C2 proteins. By contrast, cytosolic proteasomes from a homozygous cat showed elevated β-5 (chymotrypsin-like) proteolitic activity compared to wild-type or heterozygous cats. Additional experiments are necessary to determine whether aberrant protein degradation of A31P cMyBP-C contributes to disease. Supported by NIH HL080367.